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Daniel Martins Ioc Dout 2010
Daniel Martins Ioc Dout 2010
Rio de Janeiro
2010
INSTITUTO OSWALDO CRUZ
Pós-Graduação em Biologia Celular e Molecular
RIO DE JANEIRO
2010
ii
INSTITUTO OSWALDO CRUZ
Pós-Graduação em Biologia Celular e Molecular
EXAMINADORES:
REVISÃO:
Prof. Dra. Solange Lisboa de Castro – IOC-FIOCRUZ
iii
Ficha catalográfica elaborada pela
Biblioteca de Ciências Biomédicas/ ICICT / FIOCRUZ - RJ
M386
Martins, Daniel Adesse Pedra
CDD 616.9363
“É preciso estar atento e forte,
Não temos tempo de temer a morte”
(Caetano Veloso)
iv
AGRADECIMENTOS
À Diana por ser um grande amor, por me entender tão bem e por ser
sempre a melhor companhia;
v
Aos professores Herbert B. Tanowitz e David C. Spray pelo convite para
trabalhar com suas equipes em NY; pelas excelentes discussões e seus
valiosos ensinamentos; agradeço em especial a Débora, André, Mia, Márcia,
Sylvia, Fran, Vicki e Alfie pela amizade sincera começada em NY e mantida até
hoje;
Por último agradeço ao Kineret, por ser outra empreitada que me traz
tantas alegrias e realizações; por encher minha semana de música e dança e
me forçar a aproveitar e otimizar ao máximo as poucas 24 horas de cada dia.
vi
Esta tese é composta de quatro publicações, três artigos publicados em
revistas indexadas e um capítulo de livro. Outros três trabalhos foram
publicados durante o curso de Doutorado em Biologia Celular e Molecular:
vii
Resumo
RESUMO
viii
Abstract
ABSTRACT
Chagas’ disease is caused by the protozoan Trypanosoma cruzi and is the main
cause of morbidity and mortality by heart problems in endemic countries in Latin
America. In the present work we employed as experimental models cell cultures and
murine experimental infection to evaluate aspects related to the pathogenesis of this
disease. In the first part we determined through oligonucleotide microarrays the
transcriptomic signatures induced in a myoblast cell line by four reference T. cruzi
strains. We observed that the significantly altered (p<0.05) genes differed for each
strain studied and only 21 suffered changes in the same direction in all four strains.
However, infected myoblasts presented proportional alterations in the the overall
transcriptome. These results indicate that the infection with distinct strains modulate
similar, but not identical, pathways in the host cell. Next we analyzed the impact of T.
cruzi infection on cardiac caveolin-3 (Cav-3) expression, which has an important role
in the maintenance of intracellular calcium homeostasis and is also involved in
cardiac hypertrophy pathways. Both in vitro and in vivo infection induced a significant
reduction of Cav-3 levels. This reduction was followed by the activation of
extracellular signal regulated kinase (ERK), which plays a major role in cardiac
remodeling and hypertrophy, suggesting that this pathway may be involved in the
pathogenesis of Chagas’ heart disease. Finally we tested the tripanocidal potential of
the anti-arrhythmic drug amiodarone in infected cultures of cardiac myocytes. This
compound displayed a selective anti-T. cruzi activity, inducing ultrastructural
alterations in intracellular amastigotes and promoted host cell recovery with actin
cytoskeleton and gap junction reassembly, followed by restoration of the
spontaneous contractility. The data generated in this work bring important advances
into the understanding of Chagas heart disease establishment and also a new option
for the etiological treatment of this neglected disease.
ix
Lista de Abreviaturas
LISTA DE ABREVIATURAS
x
ÍNDICE
RESUMO.........................................................................................................................viii
ABSTRACT. .....................................................................................................................ix
LISTA DE ABREVIATURAS..............................................................................................x
INTRODUÇÃO..................................................................................................................1
1. A doença de Chagas..............................................................................................2
1.1 Aspectos epidemiológicos......................................................................................2
1.2 Trypanosoma cruzi - aspectos biológicos..............................................................4
1.3 Fisiopatogenia........................................................................................................6
1.4 Tratamento..........................................................................................................12
2. Interação T. cruzi-célula hospedeira.....................................................................15
2.1. Estrutura e função das cavéolas no tecido cardíaco..........................................18
2.2. Efeito da infecção sobre junções comunicantes e eletrofisiologia......................22
2.2.1 Estrutura e funcionamento das junções comunicantes.....................................23
2.2.2 Impacto da infecção na expressão de conexina43...........................................25
JUSTIFICATIVA..............................................................................................................27
OBJETIVOS....................................................................................................................29
RESULTADOS................................................................................................................30
Trabalho #1.....................................................................................................................32
Trabalho #2.....................................................................................................................42
Trabalho #3.....................................................................................................................51
Trabalho #4.....................................................................................................................70
DISCUSSÃO...................................................................................................................79
CONCLUSÕES...............................................................................................................88
REFERÊNCIAS BIBLIOGRÁFICAS................................................................................89
xii
Introdução
INTRODUÇÃO
1
Introdução
1. A doença de Chagas
2
Estágios no Triatomineo Introdução
Repasto sanguíneo (passa tripomastigota Estágios no Humano
metacíclicas pelas fezes, parasitas entram pela
ferida ou membranas mucosas) Tripomastigota metacíclico
penetram varias células no local
da picada. Dentro da célula
transformam-se em amastigota
Tripomastigota metacíclico
Amastigotas se
multiplicam por fissão
Divisões binárias
binária nas células de
tecidos infectados
Fase infectiva
Fase de diagnostico
3
Introdução
4
Introdução
1.3. Fisiopatogenia
6
Introdução
7
Introdução
Esta fase dura de um a três meses e após este período há uma diminuição na
produção de imunoglobulinas de classe IgM e aumento naquelas de classe IgG, além
de uma drástica redução na parasitemia. Neste momento há a transição para fase
crônica, na qual 20-50% dos pacientes podem permanecer em uma forma
indeterminada da doença por 10 a 20 anos e 25-35% irão desenvolver a forma cardíaca
(Dias, 2000). O diagnóstico fica limitado a métodos parasitológicos indiretos tais como
Resposta imune
Resposta imune
inadequada
adequada
Recirculação
periódica do parasita
Ativação do sistema Baixa resposta por
complemento, liberação de hipersensibilidade tardia
PDGF
Resposta por
hipersensibilidade tardia ou
autoimune
Miocardite e fibrose
Microcirculação dilatada,
lesões isquêmicas em zonas
de vascularização periférica
Alterações de matriz
extracelular
Fibrose no miocárdio Pouca inflamação, fibrose
ventricular esquerdo e ou hipertrofia do miocárdio
em nódulos SA e AV
Dilatação ventricular
Insuficiência
Arritmia Morte Súbita cardíaca crônica Forma indeterminada
9
Introdução
pesada de miosina, assim gerando uma resposta auto-imune humoral ou celular (revisto
em Kierszenbaum, 2005). Esta hipótese afastou por muitas décadas o desenvolvimento
de novas abordagens quimioterápicas específicas, inclusive para a fase crônica (revisto
em Urbina, 2010). Além disso, não foi criado nenhum programa de vacinação para
doença de Chagas, com a justificativa de que aumentando a resposta imune contra o T.
cruzi poderia causar uma maior severidade da doença auto-imune na fase crônica. A
maior evidência para a teoria da autoimunidade é a observação de que danos teciduais
são observados mesmo quando da ausência de parasitos e a presença de auto-
anticorpos no tecido cardíaco (revisto em Higuchi et al., 2003).
(b) Desenervação Parassimpática (Teoria Neurogênica): A observação de
que a infecção experimental ou humana por T. cruzi induz uma redução na população
de neurônios no tecido cardíaco, associada à inflamação periganglionar e a uma reação
autoimune antineural (Ribeiro dos Santos et al., 1979; Soares & Santos, 1999), gerou a
hipótese de que CCC estaria principalmente relacionada a danos no sistema nervoso
parassimpático. No entanto, esta hipótese perdeu força após a constatação de que a
patogênese da doença de Chagas é muito complexa e encontrou alguns obstáculos
conceituais, tais como o fato de que a CCC pode ser causada aparentemente por
qualquer cepa de T. cruzi, inclusive por aquelas não-neurotrópicas (Miles et al., 1981).
Mesmo assim a teoria neurogênica é relevante principalmente porque os danos ao
sistema parassimpático podem ser um dos mecanismos que induzem a morte súbita
nos pacientes chagásicos (revisto em Marin-Neto et al, 2007).
(c) Alterações na Microcirculação: Diversos trabalhos vêm demonstrando
que a infecção por T. cruzi, e o consequente desenvolvimento da CCC podem estar
relacionados a graves alterações na microcirculação do tecido cardíaco, incluindo
aumento da viscosidade do sangue (Berra e cols., 2005), diminuição da velocidade de
hemácias (Tanowitz et al., 1996) e aumento da agregação plaquetária (Tanowitz et al.,
1990), durante infecção experimental pelo parasito. Este efeito foi revertido com o uso
experimental de verapamil, que inibe a agregação plaquetária (Morris et al., 1989).
Baseado em investigações clínicas (Hiss et al., 2009), seria possível hipotetizar que a
hipoperfusão crônica no miocárdio pode contribuir como um mecanismo patogênico
através da geração de regiões de micro-isquemias, com o desenvolvimento de
miocitólise e fibrose reparadora no ventrículo esquerdo observadas na doença de
10
Introdução
11
Introdução
1.4. Tratamento
utilizado como quimioterápico para o tratamento anti-T. cruzi. Devido aos efeitos
colaterais causados aos pacientes a droga foi descontinuada no Brasil, Argentina, Chile
e Uruguai. Atualmente, o tratamento para doença de Chagas é feito principalmente com
o composto Benzonidazol (Rochagan® no Brasil ou Radanil® na Argentina, Roche; N-
benzil-2-nitroimidazol acetamida) (Figura 4). Esta droga atua por ligações covalentes ou
outras interações de intermediários de nitroredução com componentes do parasito ou
se ligando a DNA, lipídeos e proteínas do parasito (revisto em Coura & de Castro,
2002). Benzonidazol (Bz) tem um índice de eficácia média de 60% para casos de
infecção aguda e infecções recentes (Silva et al., 1974). Não se sabe ao certo por que
estes compostos nitro-heterocíclicos têm eficácia tão baixa, principalmente durante a
fase crônica, mas acredita-se que isto ocorra devido às propriedades farmacocinéticas
desfavoráveis e sua baixa penetração tecidual (revisto em Urbina, 2010). No entanto,
alguns estudos recentes mostraram que o uso de Bz tanto na infecção crônica
experimental (Garcia et al., 2005) e humana (Viotti et al., 2006) reduziu o parasitismo,
miocardite, alterações na condução cardíaca, extrassístole ventricular e sorologia. Estes
resultados promissores estimularam, por exemplo, a criação de um amplo estudo para
avaliar a eficácia do uso de Bz em pacientes portadores de doença de Chagas crônica
cardíaca, chamado BENEFIT (Benzonidazole Evaluation for Interrupting
Trypanosomiasis) (Marin-Neto et al., 2009), mas até o momento não há nenhum estudo
que demonstre total cura parasitológica com uso deste composto durante a fase
crônica.
Outro aspecto importante a respeito do tratamento é que existe uma importante
variabilidade biológica entre as cepas de T. cruzi em relação à virulência,
patogenicidade, tropismo tissular, estresse oxidativo e resistência à quimioterapia
(Sanchez et al., 1990; Veloso et al., 2001; Mielniczki-Pereira et al., 2007). A cepa
Colombiana, por exemplo, é destacada por sua resistência a Bz (Filardi & Brener,
1987). Por esta razão, o Programa Integrado de Doença de Chagas (PIDC), iniciativa
da Fundação Oswaldo Cruz, elaborou um protocolo para validação de novos compostos
como possíveis candidatos para tratamento de doença de Chagas, que inclui o teste in
vitro e in vivo da ação destes compostos contra cepa Y e Colombiana (Romanha et al.,
2010).
13
Introdução
15
Introdução
17
Introdução
18
Introdução
20
Introdução
Figura 5: Visão esquemática da interação entre caveolina-1 e outras proteínas, incluindo canais de
cálcio e receptor de IP3 para gerar um influxo de cálcio citoplasmático. IP3: inositol trifosfato; IP3R3:
receptor de IP3; SERCA: Ca2+ ATPase de retículo endoplasmático; NCX: Na+/Ca2+ ATPase; GPCR:
receptor ligado a proteína G; TRPC1: canal de Ca2+ acionado por voltagem; ER/SR: retículo
endoplasmático ou retículo sarcoplasmático (em células musculares); MP: Membrana Plasmática.
Traduzido de Hardin & Vallejo, 2008.
21
Introdução
camundongos Cav-1 e -3 KO, nos animais dKO Cav-1/-3 não foi possível detectar a
presença de cavéolas no sarcolema (Park et al., 2002).
Além dos danos à formação de túbulos T em cardiomiócitos pela ausência de
caveolinas, as cavéolas são abundantes em canais iônicos e transportadores tais como
o trocador Na+/Ca2+, canais de cálcio ativados por voltagem tipo L (Cav1.2), canais de
K+ e de Na+, criando complexos de sinalização celular importantes para a fisiologia da
célula (Figura 5) (Balijepalli et al., 2006; Hardin & Vallejo, 2008). Dada a variedade de
diferentes canais iônicos localizados nas cavéolas, é provável que estas contribuam
para a gênese de arritmias. Tanto as arritmias congênitas quanto as adquiridas, como
observado em pacientes com insuficiência cardíaca, podem envolver alterações nas
funções das cavéolas (revisto por Balijepalli & Kamp, 2009). Estes danos congênitos
podem incluir mutações no gene CAV3, gerando a Síndrome do QT longo tipo 9 (Vatta
et al., 2006) e a Síndrome da Morte Súbita Infantil (Cronk et al., 2007).
A importância da correta expressão de caveolinas para a fisiologia
cardiovascular fica ainda mais evidenciada com os modelos animais KO para caveolina-
1, -3 e -1/-3, uma vez que estes animais apresentaram severo espessamento da parede
do ventrículo esquerdo, fibrose intersticial, miocitólise e ativação da via de p42/44
MAPK, também chamada ERK (quinase regulada por sinal extracelular) (Woodman et
al., 2002). Esta quinase apresenta um papel importante como efetora de resposta
hipertrófica cardíaca e proliferação celular no sistema cardiovascular (Gillepsie-Brown et
al., 1995). Dados obtidos por experimentos in vitro também indicam um papel das Cav-1
e -3 como reguladores negativos da sinalização por ERK 1/2 (Engelman et al., 1998).
Durante a fase aguda da doença de Chagas em modelo murino, Nagajyothi e
colaboradores (2006) descreveram uma redução nos níveis de Cav-1, -2 e -3 e
aumento na forma fosforilada de ERK nos corações de animais infectados por T. cruzi.
Este efeito também foi observado na infecção in vitro de adipócitos, nos quais houve
redução da expressão de Cav-1 e aumento da expressão de citocinas, quimiocinas e
ativação de p38 MAPK e ERK (Nagajyothi et al., 2008). Estes dados apontam para um
papel importante desempenhado pelas caveolinas cardíacas durante a infecção por T.
cruzi.
22
Introdução
23
Introdução
Cx43 (Kanno & Saffitz, 2001). As conexinas estão distribuídas pelo coração de forma
que no ventrículo a proteína majoritária é a Cx43; no átrio e sistema de condução
(fibras de Purkinje), a Cx40 (alta condutância) e no sistema de condução
atrioventricular, a Cx45 (baixa condutância).
Durante uma injúria, o tecido cardíaco sofre remodelamento de Cx43 que em
alguns casos pode ter direta relação com o dano elétrico resultante. O remodelamento
de Cx43 pode ser dividido em duas modalidades: distribuição de placas de Cx43 e
alterações na expressão de Cx43 (proteína ou mRNA). Em zonas do miocárdio
próximas à cicatriz do infarto, há perda da ordem normal de distribuição de Cx43, cuja
imunomarcação passa a ser localizada nas laterais das fibras cardíacas. Da mesma
forma, em casos de hipertrofia cardíaca e morte súbita, é possivel observar
lateralização, desorganização e internalização de Cx43. Baixos níveis de expressão de
proteína Cx43 ou mRNA para Cx43 foram detectados em pacientes com insuficiência
cardíaca terminal, cardiomiopatia dilatada idiopática e doença cardíaca isquêmica
(Severs et al., 2004; Gourdie et al., 2006). No entanto, apenas danos que causem uma
redução de mais de 50% na quantidade de junções comunicantes no coração são
capazes de gerar efeitos na velocidade de condução (Jongsma & Wilders, 2000).
25
Introdução
após 72h não apresentaram variação nos níveis de conexina ou nos níveis de
fosforilação de Cx43 (Campos de Carvalho et al., 1998). Os autores argumentaram que
estes resultados conflitantes poderiam ser explicados por uma possível alteração no
transporte de Cx43 para a membrana plasmática ou porque a região de Cx43 que o
anticorpo reconhece estaria mascarada devido a alterações conformacionais da
proteína. Curiosamente o modelo de infecção experimental de cardiomiócitos de feto de
camundongo Swiss Webster pela cepa Y de T. cruzi (TC II) resultou no aumento da
contratilidade dos miócitos (Aprigliano et al., 1993) e em alterações nos níveis de Cx43
in vitro e in vivo (Adesse et al., 2008). Outra observação importante foi a de que o uso
de soro de pacientes chagásicos crônicos em culturas de miócitos murinos, resultou em
uma redução no acoplamento celular (Costa et al., 2000). Este resultado pode indicar
um papel de fatores solúveis presentes na cardiopatia chagásica na regulação da
expressão de conexina durante a infecção in vivo. Os níveis séricos de TGF- são
aumentados durante a doença de Chagas e estão diretamente relacionados com a
geração de resposta fibrótica no tecido cardíaco (revisto em Araújo-Jorge et al., 2008).
TGF-parece estar diretamente relacionado à redução de Cx43 observada na infecção
in vitro de cardiomiócitos de camundongo e humana por T. cruzi (Waghabi et al., 2009).
26
Justificativa
JUSTIFICATIVA
27
Justificativa
28
Objetivos
OBJETIVOS
Objetivos específicos:
29
Resultados
RESULTADOS
30
Resultados
31
Resultados
Trabalho #1:
32
Am. J. Trop. Med. Hyg., 00(0), 2010, pp. 000–000
doi:10.4269/ajtmh.2010.09-0399
Copyright © 2010 by The American Society of Tropical Medicine and Hygiene
Abstract. We examined the extent to which different Trypanosoma cruzi strains induce transcriptomic changes in
cultured L6E9 myoblasts 72 hours after infection with Brazil (TC I), Y (TC II), CL (TC II), and Tulahuen (TC II) strains.
Expression of 6,289 distinct, fully annotated unigenes was quantified with 27,000 rat oligonucleotide arrays in each of the
four replicas of all control and infected RNA samples. Considering changes greater than 1.5-fold and P values < 0.05, the
Tulahuen strain was the most disruptive to host transcriptome (17% significantly altered genes), whereas the Y strain
altered only 6% of the genes. The significantly altered genes in the infected cells were largely different among the strains,
and only 21 genes were similarly changed by all four strains. However, myoblasts infected with different strains showed
proportional overall gene-expression alterations. These results indicate that infection with different parasite strains mod-
ulates similar but not identical pathways in the host cells.
INTRODUCTION METHODS
Chagas disease, caused by infection with the flagellate pro- Cells and parasites. The L6E9 rat myoblast cell line was
tozoan parasite Trypanosoma cruzi, is a widespread disease in maintained in Dulbecco’s modified eagle medium (DMEM)
Latin America affecting millions of people.1 Infective trypo- supplemented with 10% fetal bovine serum (Invitrogen,
mastigotes invade peripheral cells and transform into multipli- Carlsbad, CA) and 1% penicillin/streptomycin at 37°C with
cative amastigote forms. The initial (acute) phase of the disease 5% CO2 atmosphere.15 Cells were dissociated with trypsin/
is characterized by intense tissue parasitism involving the ethylenediaminetetracetic acid (EDTA) solution (Gibco), and
heart, skeletal and smooth muscle cells, liver, fat, and brain that 106 cells were plated in 100-mm2 cell-culture dishes. After 24
is accompanied by intense focal inflammation and necrosis.2 hours of plating, cells were washed with Phosphate Buffered
Some patients can evolve to a chronic phase of the disease that Saline (PBS) containing Ca2+ and Mg2+ (Gibco) and infected
can include cardiac and/or digestive forms. The severity of the with 2 × 106 trypomastigote forms of T. cruzi in DMEM.
chronic phase may be related to the efficiency of the host immune Parasites of the Y, CL Brener,16 Tulahuen, and Brazil strains
response in resolving the infection during the acute phase,3 but were obtained from supernatants of infected L6E9 cultures.
this has never been proven. Moreover, there are several reports Forty-eight hours post-infection, cells were washed twice
of differences of tropism of T. cruzi to host tissue, which is also with Ca2+/Mg2+ PBS to remove free trypomastigotes in the
associated with the pathogenesis of chronic Chagas disease.4,5 supernatant, and they were re-fed with fresh supplemented
Differences in the pathogenesis of the disease among DMEM. Total RNA was harvested at 72 hours post-infection
patients may vary according to differences in both hosts and using guanidinium thiocyanate-phenol-chloroform extraction
parasite strain.6 Among differences in T. cruzi strains are their (TRIZOL) reagent (Invitrogen, Carlsbad, CA), following the
resistance to chemotherapy, oxidative stress, and infectivity in protocol indicated by the manufacturer, when at least 25% of
the mouse.7–9 Although previous in vivo and in vitro microar- the cultured cells were infected, presented only intracellular
ray analyses using cultured cells10,11 and hearts of mice12–14 amastigotes, and had no release of trypomastigotes, which
infected with T. cruzi showed that this infection results in pro- would lead to re-infection of culture.
found alterations in the host cells, the degree to which these T. cruzi genotyping. The different isolates of T. cruzi used
results are applicable to all T. cruzi strains found in infected in this work were identified using the method described
individuals has not been explored previously. by Fernandes and others17 according to their phylogenetic
Since host immune response, tissue parasitism, and para- lineage. Briefly, genomic DNA from 5 × 108 epimastigote
site strains may be important factors in the pathogenesis of forms of the Y, CL, Brazil, and Tulahuen strains was extracted
chronic Chagas disease, we have used gene-array analysis to using the DNeasy kit (Qiagen, Hilden, Germany). Multiplex
compare the alterations in host cells caused by four different polymerase chain reaction (PCR) was performed using 150
stocks of T. cruzi. The present study characterizes the transcrip- ng of DNA, and the primers were designed to recognize
tomic changes in cultured rat myoblasts that result from infec- the mini-exon gene of the parasites using a pool of five
tion with each strain and highlights common genes that were nucleotides: three were derived from a hypervariable region
similarly or differentially modulated by each strain. Analysis of the T. cruzi mini-exon repeat (T. cruzi 1 [TC1], 5¢ ACA
reveals host cell changes that might lead to an understanding CTT TCT GTG GCG CTG ATC G; TC 2, 5¢ TTG CTC GCA
of previously observed differences in pathogenesis in vivo. CAC TCG GCT GCA T; TC 3, 5¢ CCG CGW ACA ACC CCT
MAT AAA AAT G) and an oligonucleotide from a specific
region of the Trypanosoma rangelii non-transcribed spacer
* Address correspondence to Daniel Adesse, Avenida Brasil 4365,
(TR; 5¢ CCT ATT GTG ATC CCC ATC TTC G). A common
Pavilhão Carlos Chagas, Rio de Janeiro, Rio de Janeiro, Brazil. E-mail: downstream oligonucleotide corresponds to sequences present
daniel.a@ioc.fiocruz.br in the most conserved region of the mini-exon gene (ME;
1
2 ADESSE AND OTHERS
5¢ TAC CAA TAT AGT ACA GAA ACT G). PCR reaction with rat 27k oligonucleotide arrays printed by Duke University
was performed using the Multiplex PCR kit (Qiagen) with (full technical information available at http://www.ncbi.nlm
the initial denaturing cycle of 95°C (15 seconds) followed by .nih.gov/geo/query/acc.cgi?acc=GSE18175). All spots affected
30 cycles of 94°C (30 seconds, denaturing), 60°C (30 seconds, by local corruption, with saturated pixels, or with foreground
annealing), and 72°C (30 seconds, extension); a final extension fluorescence less than twice the background fluorescence
cycle of 72°C lasts for 10 minutes and is followed by a soak (where noise may obscure the quantity) were removed from the
cycle (4°C) using a PTC-100 Thermocycler (M.J. Research analysis. The background subtracted signals were normalized
Inc., Massachusetts, (USA)). PCR fragments were loaded into iteratively,19 alternating red/green, interblock, lowess and scale
a 2% agarose/ Tris base, boric acid, EDTA (TBE) gel with intraslide and interslide normalization until the fluctuation of
0.008% ethidium bromide, and images were acquired using the ratio between the spot median and the corresponding block
Kodak 1D Scientific Imaging Systems. median of valid spots became less than 5% between successive
Light microscopy. Cells (6 × 104) were plated into glass cover iteration steps. Normalized expression levels were organized
slips in 24-well plates. After 24 hours, 106 trypomastigotes were into redundancy groups (each group composed of all spots
added to cultures in fresh supplemented DMEM, and infection probing the same gene) and were represented by the weighted
was followed as described above. After 72 hours of infection, average of the values of individual spots. The abundance of
cells were washed three times and fixed with glutaraldehyde host cell transcripts was considered as significantly altered
for Giemsa staining.18 Cover slips were digitally photographed after infection if the absolute fold-change was greater than 1.5-
using a Zeiss Axioplan microscope. fold and the P value of the heteroscedastic t test (two-sample,
Microarray analysis. We used the protocol optimized in unequal variance), applied to the means of the background-
our laboratory19 according to the standards of the Microarray subtracted normalized fluorescence values in the four biological
Gene Expression Data Society. Briefly, 20 µg Trizol extracted replicas of the compared transcriptomes, was greater than 0.05.
total RNA from each culture dish was reverse transcribed in This composite criterion to identify the significantly altered gene
the presence of fluorescent Alexa Fluor 555-aha-dUTP (green expression minimizes the number of false hits without eliminating
fluorescent emission) or Alexa 647-aha-dUTP (red emission; too many true hits. The 50% change cut-off (1.5-fold) was
Invitrogen) to label cDNAs. Differently labeled RNA samples selected to be significantly larger than the overall less than 10%
from biological replicas of control (uninfected cells cultured for interslide technical noise determined for the bacterial controls.
the same duration) or infected with one strain at a time were Gene categories. GenMapp and MappFinder programs
co-hybridized (“multiple yellow” strategy20) overnight at 50°C (www.genmapp.org; Gladstone Institute, University of California,
Figure 1. Characterization of L6E9 cell infection by four T. cruzi strains. Giemsa staining of L6E9 cells after 72 hours of infection with (A) Y, (B)
CL, (C) Brazil, and (D) Tulahuen strains of T. cruzi was performed. All strains studied successfully infected the myoblasts, and at 72 hours post infec-
tion (hpi), it was possible to observe amastigote forms in the host cell’s cytoplasm as shown in details in A inset. (Bars = 50 µm.) To confirm genetic
background of the parasite stocks used for infection, genomic DNA from epimastigote forms was isolated and amplified with Multiplex PCR with
primers derived from a hypervariable region of the T. cruzi mini exon. (E) The Y, CL, and Tulahuen strains had a PCR product of 250 bp, indicating
that they belong to the T. cruzi (TC) II family, and the Brazil strain had a 200-bp product, indicating that it belongs to TC I family.
GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS 3
RESULTS
Table 1 Table 3
Y Strain Brazil strain
Gene name Gene symbol Fold change Gene name Gene symbol Fold change
Alanyl-tRNA synthetase Aars 1.85 H2A histone family, member Y H2afy 3.70
Actin, gamma, cytoplasmic 1 Actg1 1.55 Cardiomyopathy associated 3 Cmya3 3.37
Chloride intracellular channel 2 Clic2 1.55 Protein kinase C, gamma Prkcc 2.70
Cyclin-dependent kinase inhibitor 2B Myocyte enhancer factor 2D Mef2d 2.43
(p15; inhibits CDK4) Cdkn2b 1.65 Mitogen-activated protein kinase 9 Mapk9 2.12
Glycogen synthase kinase 3 beta Gsk3b 1.52 Fibroblast growth factor 21 Fgf21 1.95
Integrin alpha 1 Itga1 1.76 ATPase, H+/K+ exchanging, beta
Intercellular adhesion molecule 1 Icam1 1.79 polypeptide Atp4b 1.90
Lectin, galactose binding, soluble 3 Lgals3 1.52 Phosphatidylinositol 3-kinase,
Presenilin 2 Psen2 3.814 C2 domain containing, gamma
Protein C receptor, endothelial Procr 2.04 polypeptide Pik3c2g 1.78
Pyruvate kinase, muscle Pkm2 1.70 Paladin Pald 1.67
Syndecan -protein Sdcbp 1.854 Desmin Des 1.63
Transforming growth-factor Phosphatidylinositol 3 kinase,
beta-regulated gene 1 Tbrg1 1.73 regulatory subunit, polypeptide 3 Pik3r3 –1.53
Vascular cell-adhesion molecule 1 Vcam1 1.91 Coronin, actin-binding protein, 1B Coro1b –1.91
X-linked myotubular myopathy gene 1 Mtm1 1.53 Synaptotagmin XI Syt11 –1.91
Actin, alpha 1, skeletal muscle Acta1 –4.84 Proteasome (prosome, macropain)
Acyl-CoA synthetase long-chain family 28 subunit, beta Psme2 –2.35
member 3 Acsl3 –2.63 Keratin 25D Krt25d –2.38
ATPase, Ca++ transporting, cardiac Tropomyosin 4 Tpm4 –2.46
muscle, slow twitch 2 Atp2a2 –1.60
Cadherin 15 Cdh15 –2.88
Calsenilin, presenilin-binding protein, associated gene (3.37-fold), MMP 1B (2.44-fold), mitogen-
EF hand transcription factor Csen –1.62 activated protein kinase 9 (2.12-fold), fibroblast growth factor
Cytochrome P450, family 26, (1.94-fold), desmin (1.63-fold), and tropomyosin 4 (−2.45-fold)
subfamily b, polypeptide 1 Cyp26b1 –2.83
Guanine monphosphate synthetase Gmps –1.94
(Table 3). Ten functional categories of genes were highly
Myosin light chain, phosphorylatable, modulated in L6E9 by Brazil strain, including metallopeptidase
fast skeletal muscle Mylpf –1.68
Rho family GTPase 2 Rnd2 –1.62 Table 4
Tulahuen strain
Gene name Gene symbol Fold change
Brazil strain. The Brazil strain induced significant alteration
Activin A receptor type II-like 1 Acvrl1 7.60
of 7.31% genes of L6E9 by more than 1.5-fold. Among these 494 Cadherin 3, type 1, P-cadherin
modulated genes, 117 were decreased, and 377 were increased. (placental) Cdh3 4.65
Some interesting genes that had their transcription altered Cardiac ankyrin repeat kinase Cark 2.54
by infection with the Brazil strain were cardiomyopathy CDK5 regulatory subunit-associated
protein 1 Cdk5rap1 2.40
Chymotrypsinogen B Ctrb 2.64
Table 2 Glucose 6 phosphatase, catalytic, 3 G6pc3 1.65
CL strain Inositol 1,4,5-trisphosphate 3-kinase C Itpkc 2.37
Interleukin 11 Il11 1.68
Gene name Gene symbol Fold change
Kinesin family member C1 Kifc1 2.04
Adenylate cyclase 2 Adcy3 1.56 Myotrophin Mtpn 1.51
Alpha-spectrin 2 Spna2 1.70 ATPase, Ca++ transporting, plasma
Angiopoietin-like 2 AF159049 1.56 membrane 1 Atp2b1 –2.76
Annexin A1 Anxa1 1.56 Bcl2 modifying factor Bmf –2.93
Casein kinase 1, gamma 1 Csnk1g1 1.95 Calcium channel, voltage-dependent,
Caspase 7 Casp7 1.76 beta 3 subunit Cacnb3 –1.88
Chemokine-like factor Cklf 1.76 Calreticulin Calr –2.04
Chondroitin sulfate proteoglycan 6 Cspg6 1.64 Caspase 9 Casp9 –1.75
Desmuslin Dmn 1.60 Cytochrome c oxidase, subunit Va Cox5a –1.62
Epsin 2 Epn2 1.97 Cytokine-induced apoptosis inhibitor 1 Ciapin1 –1.67
Glutamate receptor, ionotropic, Dynein cytoplasmic 1 heavy chain 1 Dync1h1 –1.86
N-methyl D-aspartate 2B Grin2b 3.38 Ectonucleoside triphosphate
Integrin alpha 5 Itga5 1.84 diphosphohydrolase 1 Entpd1 –2.90
Interleukin 1 receptor, type I Il1r1 2.27 Farnesyltransferase, CAAX box, alpha Fnta –1.66
Matrix metallopeptidase 10 Mmp10 2.40 Hypoxia inducible factor 1, alpha subunit Hif1a –1.52
Matrix metallopeptidase 3 Mmp3 2.52 Inositol 1,4,5-triphosphate receptor 3 Itpr3 –3.70
Phosphatidylethanolamine Janus kinase 3 Jak3 –1.64
N-methyltransferase Pemt 2.03 Junctional adhesion molecule 3 Jam3 –2.25
Phospholipase D1 Pld1 1.58 Laminin, beta 2 Lamb2 –2.21
Plasminogen activator, urokinase Matrix metallopeptidase 11 Mmp11 –3.46
receptor Plaur 1.76 Muscle, skeletal, receptor tyrosine kinase Musk –2.73
Proteasome (prosome, macropain) Phospholipase D2 Pld2 –2.88
26S subunit, non-ATPase, 2 Psmd2 1.99 Protein kinase C, nu Prkcn –2.57
Chaperonin subunit 4 (delta) Cct4 –1.57 Synaptojanin 2 Synj2 –2.35
Syntaxin 8 Stx8 –1.56 Troponin T2, cardiac Tnnt2 –2.89
T-cell immunomodulatory protein Cda08 –2.61 Tumor protein p53 Tp53 –1.92
GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS 5
Table 5
Similar results
Fold change (strains)
C1q and tumor necrosis factor-related protein 1 C1qTNF1 –1.90 –1.80 –2.70 –1.80
Matrix metallopeptidase 14 MMP14 –2.00 –1.90 –2.00 –1.70
Cardiotrophin-like cytokine factor 1 Clcf1 2.20 2.30 1.80 2.30
Cut-like 1 (Drosophila) Cutl1 7.72 2.58 5.36 1.59
DNA-damage inducible transcript 3 Ddit3 9.30 3.08 2.41 2.64
Excision repair cross-complementing rodent repair deficiency,
complementation group 3 Ercc3 2.19 2.67 1.59 2.36
G protein-coupled receptor, family C, group 5, member A Gprc5a 1.94 2.53 1.87 1.99
GrpE-like 1, mitochondrial Grpel1 1.60 1.95 1.66 1.91
Pericentriolar material 1 Pcm1 2.70 4.25 2.23 3.70
Proprotein convertase subtilisin/kexin type 7 Pcsk7 6.93 2.87 2.38 2.15
Serologically defined colon cancer antigen 3 Sdccag3 3.36 2.71 2.67 2.50
Solute carrier family 1 (glutamate/neutral amino acid transporter) Slc1a4 1.59 2.50 1.82 1.68
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein Ywhaq 2.84 2.85 1.77 2.56
activity, small GTPase-mediated signal transduction, and disease biomarkers that could be useful in detecting the disease
ubiquitin cycle (Table 7). independent of the parasite strain. Only two (0.027%) were
Tulahuen strain. The Tulahuen strain of T. cruzi induced decreased, MMP-14 (−1.9-fold) and C1q and tumor necrosis
the highest percentage of altered gene expression in the factor-related protein 1 (−2.0-fold), and 11 (0.15%) increased,
myoblasts (17.35%) with 761 decreased genes and 383 increased such as solute carrier family 1 (glutamate/neutral amino acid
genes, of which, 617 and 139, respectively, were uniquely transporter; 1.9-fold), G protein-coupled receptor (2.1-fold),
observed in Tulahuen-infected dishes. Additionally, infection cardiotrophin-like cytokine factor 1 (2.13-fold), pericentriolar
with this strain significantly modulated the expression of material 1 (3.22-fold), and DNA damage-inducible transcript 3
18 gene categories, including cholesterol biosynthesis, immune (4.37-fold).Table 5 shows the list of these thirteen genes and their
response, lipid metabolism, and receptor activity (Table 7). modulation by each strain of the parasite. GenMapp analysis
Some relevant examples of host cell genes significantly modu- revealed that only nine gene categories were equally expressed
lated during infection were p-cadherin (4.64-fold), cardiac by some pairs of strains such as receptor activity (Brazil
ankyrin repeat kinase (2.54-fold), chymotrypsinogen B (2.64- and Tulahuen) and ubiquitin cycle (Brazil and CL) (Table 7).
fold), H2A histone family member Z (3.18-fold), interleukin Oppositely modulated genes. Surprisingly, our arrays
11 (1.68-fold), myotrophin (1.51-fold), caspase 9 (−1.75- revealed that some transcripts increased by one specific strain
fold), cytochrome c oxidase, subunit Va (−1.61-fold), heavy were decreased by another strain. Table 6 contains all the 24
and light chain dynein (−1.86- and −1.81-fold, respectively), genes that behaved in this manner. Some genes of interest
farnesyltransferase (−1.65-fold), hypoxia-inducible factor were cytochrome P450, family 2, subfamily d, polypeptide 22
1α subunit (−1.51-fold), janus kinase 3 (−1.64-fold), matrix (3-fold by Brazil strain and −2.9-fold by Y strain), neuropathy
metallopeptidase 11 (−3.46-fold), and cardiac troponin T2 target esterase-like 1 (2.1-fold by Y strain and −1.8-fold by CL
(−2.89-fold) (Table 4). strain), platelet-derived growth-factor receptor, β polypeptide
Genes modulated equally by all T. cruzi strains as possible (1.7-fold by CL strain and −1.6-fold by Tulahuen strain),
disease biomarkers. We observed that 13 (0.18%) host cell protein kinase D2 (1.5-fold by Brazil strain and −1.6-fold by
genes had the same pattern of significant modulation by all Y strain), and RNA polymerase 1-1 (1.6-fold by CL strain and
four strains of T. cruzi studied. These genes, thus, may represent −1.6-fold by Tulahuen strain).
Table 6
Opposite results
Fold change (strains)
Table 7
Gene categories
Gene Ontology
(GO) ID GO Name No. changed No. measured No. in GO Percent changed P value
Y strain
Correlations between infections with four strains. Our studies patterns in L6E9 cells infected by the four strains used in the
identifying individual genes that were significantly altered current investigation, we compared the entire gene-array
by the four strains of T. cruzi revealed a surprising diversity datasets obtained from the infection with each strain against
with only a few genes similarly changed by infection with all each of the other strains. With Origin software (OriginLab,
strains. However, because this analysis selects only individuals, Northampton, MA), we plotted results of these pairs as log2
it does not compare subtle global changes throughout the values of their expression ratios. In all six of the comparisons
transcriptome. To compare global trancriptomic alteration of expression changes induced by infection with separate
GENE EXPRESSION CHANGES IN T. CRUZI INFECTED MYOBLASTS: STRAIN MATTERS 7
T. cruzi strains (Figure 3), the regression coefficients (r2 values) tissue tropism.7–9 The present study was undertaken to evalu-
for these linear relations were highly significant, and P values ate the extent to which global gene-expression alteration was
in all cases were less than 0.0001. This finding indicates that similarly altered after in vitro infection of a myoblast cell line
although infection with each of the parasite strains leads to with four distinct T. cruzi strains.
only partially overlapping alterations in the genes that are The microarray analyses described in this study were per-
most affected, there is an overall similarity in the pattern of formed on the myoblast cell line L6E9 during infection with
gene-expression alterations resulting from infection with all four reference strains of T. cruzi, each with well-characterized
the strains. rates of in vivo and in vitro infectivity, resistance to che-
motherapy, and pathogenesis in vivo. We used the Y and
DISCUSSION CL strains as representatives of the TC II group of T. cruzi,
known to be found in central and eastern Brazil, which is
Chagas disease represents a spectrum of pathogenesis, commonly associated with the “mega” syndromes (cardio-
varying both in its severity and the organ systems afflicted. megaly, megacolon, and megaesophagous).22 The Tulahuen
Although various host factors, such as competence to launch strain was chosen to represent the TC I group, however, the
immune response, may account in part for differences in the Multiplex PCR performed with genomic DNA of parasites
pathogenesis of the disease, parasite strain is also an important of this strain revealed that it was actually a strain belonging
variable,6 resulting in differences in viability, infectivity, and to the TC II family. The Brazil strain was selected, because
Figure 3. Correlation of datasets obtained by microarray analyses of the infection of L6E9 cell line with Y, CL, Brazil, and Tulahuen strains of
T. cruzi. The datasets of each strain were plotted against each other in pairs of parasite strains. The histograms show how the transcriptomic changes
induced by all strains have a highly significant correlation (all have P values < 0.0001). R2 values were (A) 0.5, (B) 0.51, (C) 0.67, (D) 0.52, (E) 0.47,
and (F) 0.53.
8 ADESSE AND OTHERS
it has been shown to cause a dilated cardiomyopathy associ- Received July 17, 2009. Accepted for publication October 25, 2009.
ated with a reduction in fractional shortening and myocardial Acknowledgments: The authors thank Vicki L. Braunstein (AECOM)
wall thinning in mice.13 Genotyping revealed that the Brazil and Angela Santos (LUC–Fiocruz) for the technical support with
strain belongs to the TC I group, the predominant group in maintenance of the T. cruzi strains and Nadia Nehme and Dr. Octavio
Venezuela and central Brazil, which is usually associated Fernandes (Fiocruz) and Aisha Cordero (AECOM) for the assistance
with T. cruzi genotyping. We also thank Ethan Mackenzie (AECOM)
with electrocardiographic (ECG) abnormalities.22 for the help with GenMapp analysis. D.A. was supported in part by
We have compared the datasets obtained from the microar- a grant from the Fogarty International Center-NIH D43 W007129
ray analyses in two ways, resulting in different but comple- (HBT) and grants from The US National Institutes of Health
mentary conclusions regarding the pathogenesis of T. cruzi AI-076248(HBT), HL-73732(HBT, DCS) and from CNPq, CNPq,
PAPES IV-FIOCRUZ.
infection in vitro and perhaps, applicable to in vivo infection
as well. First, through identification of the genes that had Authors’ addresses: Daniel Adesse, Luciana Ribeiro Garzoni, and
altered expression after infection, we identified a large num- Maria de Nazareth Meirelles, Laboratório de Ultraestrutura Celular,
Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil. Dumitru
ber of gene expression changes in the infected cells, but only A. Iacobas, Sanda Iacobas, and David C. Spray, Dominick P. Purpura
a very small number of these were common to infection with Department of Neuroscience, Albert Einstein College of Medicine,
each parasite strain. We concluded from this analysis that each Bronx, NY. Herbert B. Tanowitz, Department of Pathology, Albert
strain induces a particular fingerprint of pathology. One such Einstein College of Medicine, Bronx, NY.
fingerprint may include genes encoding cell-junction proteins,
which was evidenced by our finding that both Tulahuen and REFERENCES
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Resultados
Trabalho #2
42
Report Report
Cell Cycle 9:8, 1-8; April 15, 2010; © 2010 Landes Bioscience
1
Laboratório de Ultra-Estrutura Celular; Instituto Oswaldo Cruz/FIOCRUZ; Rio de Janeiro, RJ Brazil; Departments of 2Pathology; 3Medicine; and 4the Dominick Purpura
Department of Neuroscience; Albert Einstein College of Medicine; Bronx, NY USA; 5Department of Biochemistry and Immunology; Institute of Biological Sciences; Federal
University of Mina Gerais; Belo Horizonte, Brazil; 6Department of Stem Cell Biology & Regenerative Medicine and the Kimmel Cancer Center; Thomas Jefferson University;
Philadelphia, PA USA; and 7The Muscular and Neurodegenerative Disease Unit; University of Genoa and G. Gaslini Pediatric Institute; Genoa, Italy
Caveolae are motile, membrane-bound compartments that contain a number of molecules that participate in cell
signaling. Caveolins are protein markers of caveolae and function in a variety of biological processes. Caveolin-3 (Cav-3)
is expressed in muscle cells and Cav-3 null mice display a cardiomyopathic phenotype. Ultrastructural cytochemistry,
confocal microscopy and immunoblotting revealed a reduction in Cav-3 expression and an activation of ERK (extracellular-
signal-regulated kinase) 48 hours after Trypanosoma cruzi infection of cultured cardiac myocytes. CD-1 mice infected
with the Brazil strain of T. cruzi displayed reduced expression of Cav-3 and activation of ERK 66 days post infection (dpi).
By 180 dpi there was a normalization of these values. These data suggest that the reduction in Cav-3 expression and the
activation of ERK during the early phase of infection may contribute to the pathogenesis of chagasic cardiomyopathy.
Introduction lins play critical roles in calcium homeostasis in the heart16 and
are disrupted by T. cruzi infection.17
Chagas disease is caused by the protozoan parasite Trypanosoma Caveolae are signaling platforms that compartmentalize and
cruzi,1 and results in heart disease in endemic areas of Latin concentrate signaling molecules such as G-protein subunits and
America.2 Acute infection is usually associated with intense myo- endothelial nitric oxide synthase. Caveolins inhibit the down-
cardial inflammation characterized by an upregulation of cytok- stream activation and signaling of many proteins, including c-Src,
ines and chemokines.3,4 The ensuing cardiovascular remodeling H-Ras, mitogen-activated protein (MAP) kinases, and eNOS.19,20
may result in cardiomyopathy. The dilated cardiomyopathy is Caveolin-1 (Cav-1) is expressed ubiquitously, although at differ-
associated with congestive heart failure, arrhythmias, conduction ent levels in different tissues; Cav-2 is tightly co-expressed with
abnormalities and thrombo-embolic events.2,5 The mouse model Cav-1, whereas Cav-3 is expressed predominantly in striated
of T. cruzi infection has been intensely studied because it recapit- muscle cells.21 Importantly, Cav-1 and Cav-3 null mice display a
ulates many of the pathological, functional and immunological cardiomyopathy, with left ventricle wall thickening, fibrosis and
features of the human disease. In addition, primary cultures of p42/44 MAPK (ERK 1/2) activation.22
rodent neonatal cardiac myocytes have been utilized as a tool to The activation of ERK1/2 plays an important role in the
investigate the effect of T. cruzi on the heart.6-13 For example, T. pathogenesis of cardiac hypertrophic responses and cellular pro-
cruzi alters cardiac myocyte Ca 2+ homeostasis.6-13 and gap junc- liferation in the cardiovascular system.23 In vitro data support
tional communication.14,15 These infection-associated changes a role for Cav-1 and Cav-3 as negative regulators of ERK sig-
may contribute to the arrhythmias observed in this infection. naling.24 During acute infection with the Tulahuen strain of T.
Caveolae are motile, membrane-bound compartments con- cruzi Nagajyothi et al.17 described reduced levels of Cav-1, -2 and
taining molecules that participate in cell signaling such as -3 and increased ERK phosphorylation in hearts from infected
enzymes that generate messengers from substrates in the envi- mice. T. cruzi infection of mice and of cultured endothelial
ronment, substrates that are enzymatically converted into mes- and smooth muscle cells resulted in activation of AP-1 which
sengers and high-affinity binding sites that concentrate chemical participates in cardiovascular remodeling through ERK 1/2
signals. Caveolins are protein components of caveolae that are phosphorylation.25,26
important in modulating a variety of biological functions includ- We now report that the infection of cultured cardiac myo-
ing the modifications of signaling systems.18 Caveolae and caveo- cytes with T. cruzi resulted in a reduction of Cav-3 expression
*Correspondence to: Michael P. Lisanti and Herbert B. Tanowitz; Email: michael.lisanti@kimmelcancercenter.org and herbert.tanowitz@einstein.yu.edu
Submitted: 02/08/10; Accepted: 02/10/10
Previously published online: www.landesbioscience.com/journals/cc/article/11509
extent of Ca 2+ sparks was observed in smooth muscle cells and contribute to the occurrence of arrhythmias observed in cha-
in cardiac myocytes: These observations suggest that alterations gasic heart disease.
in the molecular assembly and ultra-structure of caveolae may One major mechanism by which expression of caveolins is
lead to pathophysiological changes in Ca 2+ signaling.16 Through downregulated is activation of the mitogen-activated protein
disruption of Ca 2+ homeostasis, alteration in caveolae may kinase family (MAPK), including the p42/44 MAPK, also
known as extracellular signal regulated kinase (ERK 1/2).19,35 immunohistochemistry and immunoblot; and a 3-fold increase
The infected myocytes displayed activation of this protein kinase in ERK phosphorylation was observed. This in accordance with
starting at 2 hours post infection as determined by immunoblot previous studies that shown reduced Cav-1, Cav-2 and Cav-3
analysis using phospho-specific antibodies, without any signifi- during acute murine T. cruzi infection followed by ERK phos-
cant alteration in total protein expression. This observation is phorylation.3,17 Cav-1 null mice display hyperactivation of ERK
consistent with our previous demonstration of ERK activation in in the heart and these mice also display cardiac hypertrophy with
cultured endothelial and smooth muscle cells after infection with normal substrate utilization and expression of genes involved
the Tulahuen strain of T. cruzi.25 in energy metabolism.36-38 In addition, Cav-3 null mice display
Since ERK phosphorylation is involved in cardiac remodel- increased expression of phosphorylated ERK and a cardiomyo-
ing, we studied Cav-3 and phospho-ERK expression in heart pathic phenotype.22
lysates of infected mice 66 days and 180 days post infection. The observations in the current report demonstrate that
Cav-3 levels were drastically reduced at 66 dpi as observed by T. cruzi infection alters Cav-3 expression in cardiac myocytes.
The ERK 1/2 activation indicates that Cav-3 downregulation is mice. For mouse experiments 6–8 week old male CD-1 mice
an important contributor in the cardiac injury observed during were injected with 5 x 104 trypomastigotes of the Brazil strain
chagasic cardiomyopathy, including the hypertrophy, inflamma- and parasitemia was evaluated by counting in a hemocytometer.
tion, fibrosis and arrhythmias, as observed in Cav-1, Cav-3 and All experiments were approved by the Institutional Animal Care
the double knockout mice.22,36,37 These observations also suggest Committee of the Albert Einstein College of Medicine. For in
that the caveolins may provide a novel target for potential thera- vitro experiments the Y strain was utilized. These trypomastig-
peutic agents. otes were maintained in cultured cardiac myocytes as previously
described until used.39
Material and Methods CD-1 mice infected with the Brazil strain had a mortality of
50% by 45 days post infection (dpi). The parasitemia peaked at
Parasites and mice. The Brazil strain of T. cruzi was main- 30 to 35 dpi at 7.5 x 105 trypomastigotes/ml and then waned.
tained in C3H/Hej (Jackson Laboratories, Bar Harbor, ME) There was no mortality after 45 dpi. Hearts were collected at 66
dpi and 180 dpi. Samples were either processed for immunohis- Protease Inhibitor Cocktail was obtained from Roche Molecular
tochemistry or quick frozen in liquid nitrogen, crushed using a Biochemicals (Indianapolis, IN.) Goat anti-rabbit IgG and goat
mortar and pestle (Humboldt, Schiller Park, IL), resuspended in anti-mouse IgG HRP-labeled antibody were obtained from Santa
lysis buffer and sonicated for immunoblotting. Cruz Biotechnology (Santa Cruz, CA). Anti-BIP antibodies were
Reagents and antibodies. Trypsin was obtained from obtained from Affinity Bioreagents (Rockford, IL).
Difco Laboratories (Detroit, Michigan), Type II collagenase Experiments with cardiac myocytes. Hearts were obtained
was obtained from Worthington Biochemical Corporation from 18 day old embryos of mice and disassociated by mechani-
(Lakewood, NJ). Fetal bovine serum (FBS), L-glutamine, cal and enzymatic dissociation methods using 0.05% trypsin and
penicillin, streptomycin, CaCl2, Dulbecco’s Modified Eagle’s 0.01% collagenase in phosphate buffered saline (PBS, pH 7.2)
Medium (DMEM), RPMI, glutaraldehyde, sodium cacodylate, at 37°C, following methods previously described.5 Briefly, ven-
osmium tetroxide,acetoneand bovine serum albumin (BSA) were tricular heart muscle cells were plated on 0.02% gelatin-coated
obtained from Sigma-Aldrich (St. Louis, MO). Lanthanum glass cover slips maintained in 24-well plates for immunostaining
nitrate was obtained from Merk KGaA, (Darmstadt, Germany) assays or on plastic dishes for electron microscopy and immuno-
and Epon 812 resin from Polysciences Inc., (Warrington, PA). blotting. The cells were maintained at 37°C in 5% CO2 atmo-
Anti-caveolin-3 antibody was obtained from Affinity Bioreagents sphere in DMEM supplemented with 5% FBS, 1 mM CaCl2, 1
(Golden, CO), polyclonal goat anti-rabbit Alexa Fluor 488 mM L-glutamine, 2% chick embryo extract, 1,000 U/mL peni-
antibody was obtained from Invitrogen (Carlsbad, CA), BCA cillin and streptomycin 50 µg/ml-1 (complete Eagle medium).
Protein Assay Reagent (bicinchoninic acid) and 4'-6-Diamidino- Cardiac myocytes were infected with the Y strain trypomastig-
2-phenylindole (DAPI) from Thermo Scientific (Rockford, otes at a multiplicity of infection of 10:1.
IL). Rabbit polyclonal anti-phosphorylated ERK and total Ultra-structural cytochemistry. Uninfected and T. cruzi-
ERK were obtained from Cell Signaling (Beverly, MA) and the infected cardiac myocytes cultured in plastic culture dishes were
Trabalho #3
Caveolins and Caveolae: Role in Signaling and Disease Mechanisms. Editado por
Jasmin JF, Frank PG and Lisanti MP. Landes Bioscience. 2010
51
©2010 Copyright Landes Bioscience and Springer. Not for Distribution
CHAPTER
RECENT DEVELOPMENTS IN
THE INTERACTIONS BETWEEN
CAVEOLIN AND PATHOGENS
Abstract: The role of caveolin and caveolae in the pathogenesis of infection has only recently
been appreciated. In this chapter, we have highlighted some important new data
on the role of caveolin in infections due to bacteria, viruses and fungi but with
particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi
and Toxoplasma gondii. This is a continuing area of research and the final chapter
has not been written on this topic.
1
2 CAVEOLINS AND CAVEOLAE
INTRODUCTION
The first steps in the initiation of an infection are the attachment and entry of a
pathogen into a host cell. It has long been assumed that an understanding of these initial
events may result in new methods for control and treatment of infections. A role for
caveolae and caveolin proteins in these processes has only recently been investigated. In
this chapter, it is not our intent to review all of microbiology and describe how each and
every microorganism interacts with caveolae and caveolin proteins, but rather to focus
VIRUSES
BPV1
clathrin mediated entry, biochemical inhibitors,
shuttling from endosomes co‑localization studies,
to caveosomes caveolinl shRNA, dominant
negative cavl, 293 cells,
pseudovirions 27
HPV16
clathrin/caveolae siRNA KO of clathrin, cavl,
independent, lipid raft dynamin and tetraspanins,
independent, dynamin biochemical inhibitors,
independent, tetraspanins caveolin ‑/‑ cells, dominant
involved negative inhibitors
293TT and HELA cells,
pseudovirions 23
HPV16
clathrin mediated entry, biochemical inhibitors,
shuttling from endosomes to co‑localization studies,
caveosomes caveolinl shRNA, HaCaT
cells, pseudovirions 26
HPV16/HPV31
clathrin mediated entry biochemical inhibitors,
(HPV16), caveolar uptake dominant negative inhibitors,
(HPV31) HaCaT cells,
pseudovirions 22
HPV31
Caveolar mediated uptake, biochemical inhibitors,
Rab5 mediated shuttling to co‑localization studies,
endosome dominant negative Rab5,
HaCaT cells, pseudovirions 28
inhibition of Cav‑1 and Ras signaling attenuates Tat‑induced disruption of the tight
junction proteins.
Papillomaviruses (PVs) infect the mucosal and cutaneous stratified squamous
epithelia. These infections are associated with both benign and malignant neoplasias
and the human papillomaviruses (HPVs) cause virtually all cases of cervical cancer.14
The 8 kb, circular viral genome is encapsulated by a complex of L1 (major) and L2
(minor) structural proteins.15 Upon binding to heparan sulfate proteoglycans, the PV
capsid undergoes a series of conformational changes resulting in the N‑terminus of
cervical cells. Taken together, these findings suggest that caveolin proteins might play
a role in PV entry, but it will be necessary to sort out discrepant results and inter‑type
differences in order to adequately assess their role.
BACTERIA
PROTOZOAN PARASITES
Leishmania
Depending on the species, infection with members of the genus Leishmania may
result in cutaneous, mucocutaneous, or visceral leishmaniasis. These diseases are found
wide‑spread in tropical and sub‑tropical areas of the world and are major causes of
morbidity and mortality. Additionally, leishmaniasis is an opportunistic infection in
patients with HIV/AIDS.42
Leishmania spp. have a life cycle consisting of two stages, the promastigote and
the amastigote. The extracellular promastigote develops in the gut of the sand fly vector
until it becomes a fully virulent metacyclic promastigote. Metacyclogenesis is a process
during which surface molecules associated with virulence, such as lipophosphoglycan
(LPG) and MSP (called GP63), are modulated in their expression and/or posttranslational
modifications.43‑45 Attainment of full promastigote virulence coincides with the feeding
cycle of the insect vector.43‑45 During a blood meal, the sand fly inoculates the parasite
in the skin whereupon it is phagocytosed first by neutrophils and then by macrophages,
the ultimate host cell.46 Inside the macrophage, parasites transform from promastigotes
to amastigotes over two to five days. Thereafter, amastigotes are the only form found
in the mammalian host. Amastigote replication leads to the release of amastigotes from
infected macrophages; amastigotes in turn are taken up by non‑infected macrophages,
thus spreading the infection.47,48
Leishmania enter macrophage phagosomes that ultimately fuse with lysosomes. The
survival of Leishmania spp. in this hostile intracellular environment has been primarily
attributed to the capacity of amastigotes to withstand the phagolysosomal compartment and
INTERACTIONS BETWEEN CAVEOLIN AND PATHOGENS 7
Figure 2. Caveolin‑3 (Cav‑3) expression is diminished after Trypanosoma cruzi infection: Cardiac
myocytes were isolated from mouse embryos and infected with trypomastigotes. Confocal microscopy
showed that uninfected cultures displayed abundant Cav‑3 immunoreactivity (A), including peripheral
staining. Cav‑3 signal was reduced among highly parasitized myocytes (B) and was present predominantly
at the cell periphery. DNA staining by DAPI permitted visualization of host cell nucleus and the
amastigote kinetoplastid DNA. Bars = 20 mm. Reproduced with permission from: Adesse D et al. Cell
Cycle 2010; 9:1639‑164.81
Trypanosoma cruzi
it was of great interest that during the acute phase of T. cruzi infection a reduction in the
expression of Cav‑1, Cav‑2 and Cav‑3 was observed. This was accompanied by activation
of ERK, activator protein 1 (AP‑1), nuclear factor kappa‑light‑chain‑enhancer of activated
B cells (NF‑kB) and increased cyclin D1 expression.79,80 The change in Cav‑1 expression
in infected mice was the result in part of infection of the cardiac fibroblasts since cardiac
myocytes do not express Cav‑1. At 60 days post‑infection, which is considered the
sub‑acute/chronic phase, there was a reduction in Cav‑3 expression which normalized by
day 180 post‑infection,81 though the increase in the expression of ERK persisted. Thus,
Toxoplasma gondii
FUNGI
CONCLUSION
A growing list of pathogens, including viruses, bacteria and their associated toxins,
fungi and even prions, can interact with caveolae membrane domains.2 The intracellular
trafficking of these agents via caveolae differs dramatically from the usual route of ligands
internalized by clathrin‑mediated endocytosis. The use of caveolae for cellular entry
allows the pathogen to avoid classical endosome‑lysosome trafficking and, consequently,
avoid degradative compartments within the cell.
14 CAVEOLINS AND CAVEOLAE
ACKNOWLEDGEMENTS
The work was supported by CNPq and FAPEMIG (FSM), NIH grants AI‑ 076248
(HBT) NIH Grants AI39454 (LMW) and AI045540 (MEW and Veterans Affairs Merit
Review (MEW). Daniel Adesse was supported in part by a Fogarty Training Grant D43
TW007129 (HBT). This work was also supported in part by a Career Developmental
Award (CDA‑2) from the Departments of Veterans Affairs (NER).
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Trabalho #4
Amiodarone inhibits Trypanosoma cruzi infection and promotes cardiac cell recovery
with gap junction and cytoskeleton reassembly in vitro
70
zac00111/zac9559d11z xppws S⫽1 11/16/10 4/C Fig: 5 Art: 1129-10
zjs / DOCHEAD⫽ARTICLE zjss / DOCTOPIC⫽MECHANISMS OF ACTION: PHYSIOLOGICAL EFFECTS
FROM COVER SHEET: Editor: Edlind Article No.: T Section: Mech of Action
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2011, p. 000 Vol. 55, No. 1
0066-4804/11/$08.00⫹0 doi:10.1128/AAC.01129-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
We present the results of the first detailed study of the antiproliferative and ultrastructural effects of
amiodarone on Trypanosoma cruzi, the causative agent of Chagas’ disease. Moreover, we report the effects of
this compound on the recovery of F-actin fibrils, connexin43, and contractility in T. cruzi-infected cardiac
myocytes. Amiodarone is the most prescribed class III antiarrhythmic agent and is frequently used for the
symptomatic treatment of Chagas’ disease patients with cardiac compromise. In addition, recent studies
identified its antifungal and antiprotozoal activities, which take place through Ca2ⴙ homeostasis disruption
and ergosterol biosynthesis blockage. We tested different concentrations of amiodarone (2.5 to 10 M) on
infected primary cultures of heart muscle cells and observed a dose- and time-dependent effect on growth of the
clinically relevant intracellular amastigote form of T. cruzi. Ultrastructural analyses revealed that amiodarone
had a profound effect on intracellular amastigotes, including mitochondrial swelling and disorganization of
reservosomes and the kinetoplast and a blockade of amastigote-trypomastigote differentiation. Amiodarone
showed no toxic effects on host cells, which recovered their F-actin fibrillar organization, connexin43 distri-
bution, and spontaneous contractility concomitant with the drug-induced eradication of the intracellular
parasites. Amiodarone is, therefore, a promising compound for the development of new drugs against T. cruzi.
Fn2 Chagas’ disease is the largest parasitic disease burden and a thione reductase and trypanothione synthase, and (iii) the in-
major cause of heart disease and heart-related deaths in Latin hibitors of de novo sterol biosynthesis pathways, such as imi-
America, where it affects approximately 16 to 18 million people dazole and triazole derivatives (5, 34, 35). There is also strong
AQ: B (25, 38). The disease is caused by the protozoan parasite evidence that bisphosphonates can accumulate in the parasite’s
Trypanosoma cruzi, which possesses a life cycle involving a acidocalcisomes and interfere with the activity of enzymes in-
mammalian host and an insect vector (31). volved in isoprenoid biosynthesis, such as farnesyl diphosphate
Chemotherapy against T. cruzi is limited to two compounds, synthase (13). Using T. cruzi-infected cardiomyocytes, we pre-
namely, benznidazole (a 2-nitroimidazole) and nifurtimox (a viously demonstrated that the bisphosphonate risedronate has
5-nitrofuran), which are mostly active in acute- and early a potent and selective in vitro effect against this parasite, re-
chronic-phase patients but are of limited efficacy in the prev- sulting in recovery of the cardiac cells after the treatment (16).
alent chronic stage (8, 34). Moreover, T. cruzi exhibits consid- This compound also exhibited marked in vivo antiparasitic
erable biological variability, indicating possible variations in activity in a murine model of acute Chagas’ disease (18). An-
virulence, pathogenicity, oxidative stress, and drug resistance other promising approach is the recent discovery of the anti-T.
(23, 27, 36), which may pose an important challenge in the cruzi activity of the antiarrhythmic drug amiodarone, which is
search of safer and more effective chemotherapeutic agents for frequently prescribed for the symptomatic treatment of Cha-
the specific treatment of Chagas’ disease. gas’ disease patients (4). In the heart, the effects of this com-
Studies in the last 2 decades have permitted the identifica- pound include inhibition of Na⫹ channels, L-type Ca2⫹ chan-
tion of several new drug targets for this parasite. Among the nels, K⫹ channels, and the Na⫹/Ca2⫹ exchanger, leading to its
most promising are (i) the essential cathepsin L-like protease characteristic antiarrhythmic action. It was found that the in
cruzipain, (ii) the unique Kinetoplastida enzymes trypano- vitro and in vivo activity against T. cruzi was mediated by dis-
ruption of the parasite’s Ca2⫹ homeostasis and a blockade of
ergosterol biosynthesis at the level of oxidosqualene cy-
* Corresponding author. Mailing address for Luciana R. Garzoni: clase (4).
Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz,
FIOCRUZ, Avenida Brasil 4365, Pavilhão Carlos Chagas, 2° andar, Although the antiparasitic activity of amiodarone has been
21040-900 Rio de Janeiro, Brazil. Phone: 55 (21) 2598-4535. Fax: 55 demonstrated previously, there is a lack of data regarding the
(21) 2260-4434. E-mail: largarz@ioc.fiocruz.br. Present address for effect of this compound on the ultrastructure of T. cruzi and its
Julio A. Urbina: 734 Springhill Lane, Cincinnati, OH 45226. Phone: host cells and the recovery of these cells after the antiparasitic
AQ: E (513) 321-2981. Fax: (●●●) ●●●-●●●●. E-mail: jurbina@mac.com.
† D.A. and E.M.A. equally contributed to this work and should be
treatment. Primary cultures of murine cardiac myocytes have
considered first coauthors. been the method of choice to demonstrate alterations in the
䌤
Published ahead of print on ●●●●●●●●. host cell induced by this parasite. In these studies, many as-
1
AUTHOR: Publication of this article cannot proceed without the signature
of the person who read and corrected the proof on behalf of all the authors: signature date
zac00111/zac9559d11z xppws S⫽1 11/16/10 4/C Fig: 5 Art: 1129-10
FIG. 2. Time and concentration dependence of the effects of amiodarone on infection of HMCs by T. cruzi (protocol ii). (A to C) Amiodarone
(5 to 10 M) was added to the cultures after 48 h of infection, and the percentage of infected cells (A), number of intracellular parasites per host
cell (B), and number of released trypomastigotes in the supernatant (C) were followed as a function of time. (D) We did not observe significant
cytotoxic effects of amiodarone with the concentrations used throughout the experiments. Asterisks indicate statistical differences in relation to
control cultures. The graphs show the means and standard deviations from triplicates of one representative experiment of four independent
experiments.
ductions in the percentages of infected cells were observed. apparent cytotoxic effects on the myocytes, as shown in Fig. 1C
The inhibition was statistically significant after 24 h of treat- and 2D.
ment with 10 M amiodarone (P ⬍ 0.05), 48 h with 5 M (P ⬍ Light microscopy observations showed that treatment with
0.01), and 96 h with 2.5 M (P ⬍ 0.01) (Fig. 1A). IC50s with amiodarone induced drastic morphological alterations on in-
protocol i were 5.85 ⫾ 1.4 M at 96 h and 3.14 ⫾ 1.2 M at tracellular amastigotes (Fig. 3B, C, and E, insets). We con-
120 h. Amiodarone had also an inhibitory effect on the number firmed this observation through transmission electron micros-
of intracellular amastigotes (Fig. 1B), as revealed by light mi- copy of infected HMCs and released parasites after 144 h of
croscopy (Fig. 3), significant for 10 M amiodarone at 24 h of treatment of infected HMCs with 5 M amiodarone. Infected
treatment (P ⬍ 0.05). Addition of amiodarone to cultures at untreated HMCs displayed abundant intracellular amastigotes
48 h after infection (protocol ii) also led to a highly significant in the cells’ cytoplasm, with the expected kinetoplast morphol-
reduction in the percentage of infected cells (Fig. 2). After 48 h ogy (bar shaped). A drastic loss of cytoplasmatic content and
of treatment (96 h of infection), 5 M amiodarone already the formation of membrane inclusions inside the amastigotes
reduced significantly (P ⬍ 0.05) the parasitism of cultures, were visualized in treated cultures; these cells also exhibited
which was also observed at 96 h treatment with 2.5 M ami- mitochondrial swelling, disorganization of reservosomes and
odarone (Fig. 2A). In this protocol, IC50s were 4.47 ⫾ 0.3 M the kinetoplast, probably associated with the disruption of
and 2.24 ⫾ 0.24 M at 96 and 120 h, respectively. This inhib- Ca2⫹ homeostasis (4), and a blockade of amastigote-trypomas-
itory effect was also observed for the number of intracellular tigote differentiation. Untreated amastigotes spontaneously re-
amastigotes in the HMCs (Fig. 2B). Moreover, amiodarone leased from their host cells also displayed their characteristic
inhibited the release of trypomastigotes from infected cells in morphology (Fig. 4C). There was a marked damage in amas- F4
this model after completion of the intracellular cycle of the tigotes released to the medium from amiodarone-treated (5 AQ: C
parasite. In control (untreated)-infected HMC cultures, the M) cultures, such as kinetoplast alteration, and in the Golgi
first two peaks of trypomastigote release to the supernatant apparatus (Fig. 4D).
occurred at 96 h and 192 h postinfection (Fig. 2C). Treatment Cell physiology recovery after treatment with amiodarone.
with 5 and 10 M amiodarone, starting 48 h postinfection, In order to assess the recovery of host cell ultrastructure and
drastically inhibited such release after 48 h of treatment (96 h physiology after the treatment of infected cultures with amio-
postinfection), while at 96 and 144 h, no trypomastigotes were darone, we evaluated gap junction protein Cx43 (detected by
detected in the supernatant (Fig. 2C). Amiodarone at the con- immunofluorescence) and actin filaments (stained with phal-
centrations used in the experiments (up to 10 M) had no loidin). T. cruzi infection is known to disrupt gap junctional
zac00111/zac9559d11z xppws S⫽1 11/16/10 4/C Fig: 5 Art: 1129-10
FIG. 3. Effect of amiodarone on the intracellular cycle of T. cruzi. Primary cardiac myocytes were obtained and infected with the Y strain of
T. cruzi. After 2 h of infection, cultures were treated with 5 or 10 M amiodarone. At 96 h postinfection (hpi), untreated cultures (A) displayed
cells with trypomastigote forms of the parasite already evading host cells. Cultures treated with 5 M (B) and 10 M (C) of amiodarone displayed
intracellular parasites with severe morphological alterations (arrows). (C) Interestingly, an amiodarone concentration of 10 M induced a drastic
reduction of parasitism. After 144 h of infection (corresponding to 142 h of treatment), we observed that in nontreated cultures (D), 70 to 80%
of cardiomyocytes were infected. Treatment with 5 M amiodarone (E) drastically reduced the number of parasites, whereas the use of 10 M
amiodarone (F) almost eliminated the parasitism of the cultures. Insets show details of intracellular parasites in higher magnification. P, parasites;
N, nucleus.
F5 communication through Cx43 protein reduction (1). Figure 5 fibrils, as revealed by phalloidin staining. After treatment with
shows that infected untreated HMCs (Fig. 5D to F) totally lost 5 M amiodarone for 144 h, infection was nearly abolished, as
Cx43 immunoreactivity at 192 h postinfection compared to that revealed by DAPI staining. In these cultures, Cx43 levels were
of control uninfected cells (Fig. 5A to C), which displayed comparable to those of age-matched uninfected cultures (Fig.
well-formed gap junction plaques (Fig. 5C) and abundant myo- 5I), and phalloidin staining revealed the presence of both po-
zac00111/zac9559d11z xppws S⫽1 11/16/10 4/C Fig: 5 Art: 1129-10
FIG. 4. (A) Effects of amiodarone on the ultrastructure of intracellular T. cruzi (Y strain) amastigotes. Untreated HMCs after 192 h of infection
with T. cruzi, displaying severe intracellular damage and intact amastigote forms (P) and presenting a bar-shaped kinetoplast (K), nucleus (N), and
flagellum (F). (B) Infected cultures treated with 5 M amiodarone for 144 h showed parasites with membrane blebs (arrows), loss of intracellular
material (stars), and kinetoplast (K) alterations. (C) Released parasites from untreated cultures displayed acidocalcisomes (AC) and a bar-shaped
kinetoplast (K), reservosome (R), and flagellum (F), as expected. (D) Parasites obtained from cultures treated with 5 M amiodarone (144 h)
showed important alterations in the kinetoplast and Golgi apparatus (arrow).
lygonal and filamentous structures, indicative of cytoskeleton paratus as well as the disappearance of the gap junctions of
reassembly (Fig. 5H). these cells. After 48, 96, and 144 hours of infection (protocol i),
We also assessed the impact of T. cruzi infection on cardiac untreated controls displayed a 28, 31, and 45% reduction in
myocyte spontaneous electrocontractility, and the results are spontaneous contractility (P ⬍ 0.05), respectively, whereas
shown in Fig. 5J. The parasites’ proliferation was associated treatment with amiodarone (5 M) restored the number of
with a progressive reduction in the cardiomyocytes’ contrac- spontaneous beatings to levels indistinguishable from those of
tion, which followed the disorganization of the contractile ap- uninfected cultures at 96 (P ⬍ 0.05) and 144 (P ⬍ 0.01) hours
zac00111/zac9559d11z xppws S⫽1 11/16/10 4/C Fig: 5 Art: 1129-10
4
C
O
L
O
R
FIG. 5. Amiodarone induced the recovery of host cell homeostasis. After eradicating the infection, we observed the recovery of morphological
and functional aspects of cardiac myocytes cultures, as assessed by immunofluorescence and measuring of the spontaneous electrocontractility of
the cells. (A to C) Uninfected cultured myocytes displayed abundant connexin43 immunoreactivity (C) as well as striated patterns of F-actin
staining (B). (D to F) Highly infected cultures, at 144 h of infection, presented destruction of the F-actin cytoskeleton (E) and loss of Cx43 plaques
(F). (G to I) Treatment with 5 M amiodarone for 142 h decreased parasitism and induced recovery of host cells, as evidenced by F-actin and Cx43
recovery, which included striations (H) and the presence of gap junction plaques (I). (J) The spontaneous electrocontractility of the myocytes
culture was evaluated by counting the number of contractions per 10 s. We observed that T. cruzi-infected cultures had a progressive decrease in
electrocontractility, whereas treatment with 5 and 10 M amiodarone restored cultures to normal levels of electrocontractility. *, P ⬍ 0.05; **,
P ⬍ 0.01 (ANOVA).
zac00111/zac9559d11z xppws S⫽1 11/16/10 4/C Fig: 5 Art: 1129-10
of treatment. We observed no significant effect on contractility evidence that the infection affects Cx43 translation and/or its
when the same dose of amiodarone was added to uninfected subsequent trafficking to the plasma membrane, possibly due
cultures (not shown). to microtubular damage (24). After the amiodarone-induced
disappearance of the intracellular parasites, the host cells were
capable of synthesizing new Cx43 molecules and made their
DISCUSSION
successful delivery to the plasma membrane. More impor-
Human Chagas’ disease results from infection by T. cruzi, tantly, HMCs restored their spontaneous contractility after
and tissue damage arises from both direct parasite action and treatment with 5 M amiodarone to levels comparable to
the inflammatory process that ensues (7). Cytolysis and fibrosis those in control cultures. This observation is of interest since it
are key components associated with Chagas’ disease’s patho- permits us to speculate that during in vivo infection, T. cruzi
logical manifestations, along with a sustained and diffuse in- disturbs synchronous contractility, which can be reverted with
flammation of the affected organs (31). The growing percep- the use of amiodarone alone or in combination with another
tion that chronic-phase manifestations are associated with the inhibitor of the parasite’s sterol biosynthesis, such as posacon-
persistence of the parasite in the mammalian tissues (32) and azole, which results in synergistic effects (4).
that the two available drugs (benznidazole and nifurtimox) To summarize, our results showed that amiodarone has a
have important limitations, particularly in the chronic phase selective antiproliferative effect on T. cruzi in an in vitro model
(6), has stimulated the search for new trypanocidal com- of infection of cardiac cells. Treatment induced ultrastructural
pounds. Amiodarone is frequently used as an antiarrhythmic in damage to intracellular amastigotes but promoted full struc-
chronic-phase Chagas’ disease patients with cardiac compro- tural and functional recovery of the host cells. This compound
mise (21, 26), and it also has antifungal (9, 10) and antiproto- should be considered, beyond its known antiarrhythmic activ-
zoal (4, 28) activities, which were recently described. It was ity, as an important antiparasitic agent and a lead for the
shown that the drug disrupts the parasite’s Ca2⫹ homeostasis development of new specific treatments of this neglected dis-
and also blocks ergosterol biosynthesis, resulting in excellent ease.
parasiticidal activity with low cytotoxicity (4). The results ob-
tained in this work clearly confirm that amiodarone has potent ACKNOWLEDGMENTS
and selective activity against T. cruzi, with no significant effects This work was supported with grants from FIOCRUZ, CNPq,
on their preferred host cells, cardiomyocytes, and at doses that FAPERJ, and the Howard Hughes Medical Institute (grant 55000620
do not induce the well-known antiarrhythmic action of the to J.A.U.), in part by the U.S. Public Health Service (NIH grant GM
drug on these cells. This selective action was confirmed by 65307 to E.O. and R.D. and NIH NRSA grant GM 65782 to G.L.M.),
and by the American Heart Association, Midwest Affiliate (supporting
ultrastructural analysis of amiodarone-treated cultures (Fig. E.O.) and National Center (supporting R.D.). D.A. was in part sup-
4), which revealed massive alterations of the parasites, allowing ported by grant NIH D43 W007129 from the Fogarty International
at the same time the full structural and functional recovery of Center. AQ: D
the cardiomyocytes (see below). The ultrastructural effects
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Discussão
DISCUSSÃO
79
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Discussão
Burleigh, 2003), ainda não foi elucidada a participação da via ativada por cardiotrofina
durante a infecção por T. cruzi. No entanto, a redução na expressão deste transcrito
observada em nossos experimentos sugere que a ativação de cardiotrofina-1 descrita
por Chandrasekar e colaboradores (1998) pode ser proveniente de macrófagos
ativados (Harris et al., 2006), presentes em infiltrados inflamatórios.
Outra observação interessante foi a de que diversos genes que codificam
proteínas juncionais foram afetados pela infecção por T. cruzi, incluindo plakoglobin,
junctional adhesion molecule 3, e adipocyte-specific adhesion molecule, bem como
cadherin 15 (pela cepa Y). Nosso grupo já demonstrou que a infecção de cardiomiócitos
por T. cruzi altera a expressão de moléculas de adesão como Cx43 (Adesse et al.,
2008) e caderina/catenina (de Melo et al., 2008). Como demonstrado num modelo
murino de sepse (Celes et al., 2007), alguns tipos de cardiomiopatias apresentam
alterações ou mutações em genes relacionados a proteínas do disco intercalar,
sugerindo que estas doenças, possivelmente incluindo a CCC, são “doenças juncionais”
(revisto por Spray & Tanowitz, 2007).
O segundo tipo de análise que realizamos em nossos estudos de microarray foi
uma comparação global entre os transcriptomas dos mioblastos infectados com cada
cepa, considerando inclusive as pequenas alterações (ou seja, menores que 1,5 vezes).
Nestas análises, comparamos os dados obtidos com cada cepa com aqueles obtidos
com as outras três cepas, gerando seis combinações altamente correlacionadas
(p<0,001). Esta abordagem enfatiza o conceito de que apesar da doença de Chagas
apresentar um amplo espectro de manifestações in vivo, na realidade representa uma
síndrome de alterações fenotípicas em comum, como revelado por nossos resultados.
Os genes comumente alterados pelas quatro cepas estudadas podem servir como
biomarcadores de doença aguda em áreas endêmicas, uma vez que tais cepas são
usadas como referência para diversos estudos (Gruson & Borodovitz, 2010).
Para compreender melhor o impacto da infecção por T. cruzi na fisiologia da
célula hospedeira, estudamos a expressão de Cav-3 em cardiomiócitos infectados in
vitro e no coração de camundongos durante infecção in vivo. Este estudo foi motivado
por observações prévias de nosso laboratório, que mostraram alterações na
homeostase de cálcio de cardiomiócitos durante infecção in vitro (Garzoni et al., 2003),
que incluía a redução de sítios de cálcio de superfície e cavéolas das células
83
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84
Discussão
85
Discussão
87
Conclusões
CONCLUSÕES
88
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